Study On The Relationship Between Larval Development And Signaling Molecules For Amphibalanus Amphitrite

Marine biofouling is a problem for humans in the development and utilization of marine resources,in which the marine organisims gather and grow on the artificial surfaces.With the development of ocean exploitation,the problem of biofouling has also received widespread attention.In recent years,the development of new non-toxic,highly-efficient,and environment-friendly anti-fouling technologies has become an inevitable trend of marine anti-fouling.The exploration for the mechanism of fouling organisms' attachment is the basic condition for the development of new technologies.Exploring the attachment mechanism of fouling organisms is the basic condition for the development of new technologies.Marine fouling organisms include animals,plants and microorganisms.Animals mainly refer to marine invertebrates.Barnacles are one of the most common marine fouling organisms because of their superior tolerance and wide distribution.The barnacle used in this study is Amphibalanus amphitrite.The life history of the A.amphitrite can be divided into the planktonic life stage and the fixation life stage.The process of shifting from plankton to fixation is the focus of our research.In this paper,a model of adult domestication and larval rearing was established,and the changes of the morphological characteristics during the larval development and metamorphosis were examined.Previous studies have shown that histamines are involved in the signal transduction 'during the metamorphosis of barnacle larvae[116,117].In this study,the histamine content was quantitatively determined,a series of indicators such as neurotransmitters,second messengers,and protein kinases that may be involved in the histamine signaling process were examined to investigate the unclarified histamine signal transduction pathways.The detailed results of this study were as following.(1)For adult of barnacle A.Amphitrite,the laboratory domestication needs to ensure the water temperature above 20 ?,the low temperature or insufficient nutrition will affect the release of their larvae.The barnacle larvae need to be cultured in laboratory for about 5 days before they can be metamorphosed into cyprids.During the culture process,regular microscope examinations are needed to observe the growth and development.The experimental results showed that the body shapes of barnacle larvae had a great change in the process of development and metamorphosis,which provides indicators for us to determine the various stages of the barnacle larvae.At certain stages,certain specific structures of the barnacle larvae develop rapidly.For example,their shell sizes almost double after the larvae become cyprids.(2)According to the results of Qiu[115],histamine promotes the development and metamorphosis of the larvae of barnacle of A.Amphitrite.Therefore,histamine and three other common neurotransmitters,including dopamine,epinephrine,and serotonin,were examined in this study.In order to explore how the signal molecules participates in the process of the attachment and metamorphosis of the barnacle larvae,it is first necessary to confirm whether the signal molecules are present in the larvae,followed by quantitative analysis.The results of this study show that it was failed to detect dopamine,epinephrine,and serotonin in the larvae of barnacle of A.Amphitrite by HPLC,which may be due to the absence or that the content is lower than the detection limitation.The presence of histamine was successfully detected,and quantitative analysis revealed significant differences in histamine content in the larvae of different stages.The changing trend concluded that the development and metamorphosis of the barnacle larvae consumes histamine in the body.(3)During the development and metamorphosis of barnacle larvae,there was a significant change in RNA and DNA content for the larvae of different stages.Both trends of RNA and DNA content in terms of per protein content are consistent with the change trend of histamine content,in which the RNA is more consistent than DNA.Compared with the result of histamine content,the perfect correlation was observed between the content change of RNA and histamine in the whole process of larval development and metamorphosis of barnacle of A.Amphitrite,thus verifying that histamine participated in the signal transduction process of the development and metamorphosis of barnacle larvae.(4)PKA and PKC are important protein kinases during signal transduction.During the whole larval development and metamorphosis of barnacle of A.Amphitrite,the changes of expression levels(per protein)of PKA and PKC are very similar.At the same time,the similar tendency was observed between the RNA content and expression levels of PKA and PKC.The RNA content(per protein)in nauplius IV is 1.51 times of that in nauplius V.To some extent,it is verified that the increased RNA content in the larvae led to the increased protein synthesis,including PKA and PKC expression.(5)In the process of signal transduction,the main function of second messengers is to conduct and amplify the signal in the cells.The four kinds of them examined in this study as following:DAG,cGMP,cAMP and IP3.It was found that there was a huge difference in the second messenger content in the larvae of different stages.Among them,the content of DAG is the lowest,and the content of cGMP and IP3 is the highest.There was a significant difference in the content of the same second messenger in the larvae at different stages.In addition,compared with the change trend of histamine content,the perfect correlation was observed between the contents of IP3 and histamine.Therefore,it is hypothesized that IP3 may be the second messenger involved in the process of histamine signaling during barnacle larval development and metamorphosis,and the PIP2 pathway may be involved in the signaling pathway.In summary,the trends of PKA and PKC expression were also consistent with the content trends of RNA and histamine,but the results of the second messengers detection showed that cAMP and DAG should not be the downstream carriers of histamine signal.Therefore,PKA and PKC may not be very closely related to histamine signaling,but may be related to the signaling of larval development and metamorphosis in barnacle of A.Amphitrite.All in all,the regulatory mechanisms for the signaling in the larval development and metamorphosis in barnacle of A.Amphitrite are very complex,and most likely include a combination of multiple signaling pathways.